PROJECT SUMMARY/ABSTRACT Germinal centers (GCs) are the site of antibody affinity maturation, and as such are essential to the establishment of a protective immune response. Affinity maturation is the consequence of somatic hypermutation (SHM) of the B cell receptor, followed by positive selection of B cells that acquired affinity- enhancing mutations, likely though their interaction with GC-resident T follicular helper (Tfh) CD4+ T cells. In recent years, a population of Foxp3+ Treg cells that expresses the same cell surface markers as Tfh cells (CXCR5 and PD-1), referred to as T follicular regulatory (Tfr) cells, has emerged as a potential regulator of the GC reaction. However, despite intensive study of this particular population, the precise role of Tfr in GC selection remains unclear. While most studies to date focus on potential effects of this population on preventing the emergence of autoreactive cells as a byproduct of SHM, these studies have so far failed to reveal strong autoimmune phenotypes associated with loss of this population, beyond their ability to inhibit T cell help to B cells in vitro. On the other hand, at least one study has hinted at a potential role for Tfr in controlling the stringency of the selection of high-affinity B cells in the GC, and another study has suggested based on computational simulations that Treg may be particularly effective at terminating the GC reaction. It is this latter aspect of Treg biology that we wish to address, given its potential impact on our ability to control the specificity and output of the GC reaction and consequently impact our ability to vaccinate against recalcitrant pathogens such as HIV, Influenza, and Plasmodium. In this application, we argue that the shortcomings in our current understanding of Tfr function stem primarily from unresolved issues regarding the origin, antigen- specificity, and dynamic behavior of GC-resident Tregs. We propose to use advanced multiphoton imaging and sequencing techniques to investigate these aspects of GC Treg biology, with the goal of setting up the fundamental knowledge that will be required for further functional studies.